Chronic hypoxia (CH) associated with obstructive lung diseases and sleep apnea leads to pulmonary hypertension. Elevated basal vascular smooth muscle (VSM) tone and enhanced vasoconstrictor reactivity play a prominent role in mediating CH-induced pulmonary hypertension. However, the mechanisms by which CH elicits these vasoconstrictor responses are not understood. Pilot studies have revealed a novel effect of CH to promote myogenic tone and enhance vasoconstrictor reactivity through a RhoA/Rho kinase-dependent Ca2+-sensitization pathway in pulmonary VSM. These studies further suggest that CH-induced VSM membrane depolarization stimulates RhoA/Rho kinase independent of changes in VSM free Ca2+ levels, and provide intriguing evidence for a major contribution of NADPH oxidase (NOX)-derived reactive oxygen species (ROS) to this response. Therefore, the proposed studies will investigate the central hypothesis that CH- induced VSM membrane depolarization mediates NOX-dependent stimulation the RhoA/Rho kinase pathway leading to increased myogenic and agonist-induced myofilament Ca2+ sensitivity. This hypothesis will be addressed by the following specific aims: Specific Aim #1: Identify the intracellular signaling mechanism responsible for elevated myogenic and agonist-dependent constriction following CH. We hypothesize that CH augments pulmonary arterial myogenic tone and agonist-mediated constriction via RhoA/ROK-induced VSM Ca2+ sensitization. Specific Aim #2: Determine the contribution of membrane depolarization to increased basal and agonist-induced pulmonary arterial constriction following CH. Based on pilot data outlined below, the working hypothesis for this aim is that CH-induced VSM membrane depolarization stimulates the RhoA/Rho kinase pathway leading to increased basal and agonist-induced myofilament Ca2+ sensitivity. Specific Aim #3: Establish the role of NOX-derived ROS in mediating enhanced myogenic and agonist-dependent pulmonary vasoconstrictor reactivity following CH. We hypothesize that depolarization-induced ROS-generation by NOX upregulates basal and agonist-induced RhoA/Rho kinase signaling in pulmonary VSM following CH. The proposed research will employ whole animal, isolated lung, isolated vessel and molecular approaches to establish a novel mechanistic link between VSM membrane depolarization, NOX-derived ROS generation, and RhoA/ROK signaling in the hypertensive pulmonary circulation. Collectively, these outcomes are expected to fundamentally advance our understanding of signaling mechanisms by which ROK regulates vascular tone, as well as vasoconstrictor mechanisms that contribute to the development of pulmonary hypertension. PUBLIC HEALTH RELEVANCE: Chronic obstructive pulmonary diseases (COPD) such as emphysema and chronic bronchitis are established major causes of mortality and morbidity in the U.S. COPD is currently the fourth leading cause of death in the U.S. and is predicted to be the third leading cause of death by 2020 (Sin, D.D. and Man, S.F. Proc Am Thorac Soc 2:8-11, 2005). The aim of this study is to determine the factors responsible for the development of pulmonary hypertension in a rat model of COPD. Such disease states cause poor oxygenation of the blood (hypoxia), and the resultant pulmonary hypertension leads to right heart failure and mortality in humans.